Iron dextran was developed for the treatment of iron deficiency conditions, and originally was administered by intramuscular injection to iron deficiency anemia patients who could not tolerate various formulations of oral iron salts. See, e.g., Lawrence, “Development and Comparison of Iron Dextran Products,” PDA Journal of Pharmaceutical Science & Technology 52(5):190-197 (1998). Subsequently, iron dextran was also administered intravenously and found to produce a similar beneficial outcome.
A variety of iron-containing formulations have been developed. Intravenous injections of colloidal ferric hydroxide preparations, particularly iron sucrose, are clinically indicated for the treatment of iron deficiency anemia in patients undergoing chronic hemodialysis who are receiving supplemental erythropoietin therapy.
Iron sucrose is formulated as a colloidal suspension and administered as a prodrug that is taken up by cells of the reticuloendothelial system, which release ionic iron. The ionic iron binds to transferrin, which, in turn, transfers it to the bone marrow for erythropoiesis or to ferritin and the iron storage pool in the marrow, spleen and liver.
Physiology and Metabolism of Iron
The human body stores trivalent iron as ferritin and hemosiderin. Ferritin is comprised of an outer protein shell containing a storage cavity for a polynuclear ferric hydroxide/phosphate core of approximate composition [(FeOOH)8(Fe)—OPO3H2)]n. Ferritin's protein shell, apoferritin, is composed of 24 polypeptide subunits forming the apoferritin molecule, which has an average molecular weight of about 440,000. Apoferritin's outer shell has a diameter of approximately 13 nanometers (130 Å) with an interior cavity of about 7 nanometers (70 Å).
Ferritin's protein shell, apoferritin, functions as a ferroxidase enzyme in the binding and oxidizing of divalent iron which it then stores within its cavity as a polynuclear ferric hydroxide/phosphate core. Ferritin may contain up to 4,500 polymerized ferric ions with a molecular weight for the entire molecule ranging from 700,000 to 800,000. Over 30% of the weight of the ferritin molecule may be iron.
When the amount of available iron exceeds ferritin's iron storage mechanism, an aggregated ferritin is formed called hemosiderin, which is a normal constituent of the monocyte-macrophage system. Hemosiderin is composed of molecules of ferritin, which have lost part of their protein shell and become aggregated. Hemosiderin accounts for about one third of normal iron stores and accumulates as insoluble granules in the cells of the reticuloendothelial system.
Ferritin is water soluble and may enter the blood stream through osmosis. Normal serum levels of ferritin are dependent on sex/age and range between 40 and 160 ng/mL. It is believed that in the blood stream, ferritin slowly releases divalent iron in conjunction with a reducing agent, such as reduced flavin mononucleotide and to a lesser extent, ascorbic acid. The divalent iron is oxidized back to trivalent iron by ceruloplasmin, then tightly bound to the blood protein apotransferrin forming transferrin. The molecular weight of transferrin is about 76,000 and each molecule has two binding sites for ferric ions.
Upon administration to a patient, an iron sucrose complex (or other trivalent iron colloids formulated, e.g., with gluconate, dextran, sorbitol or dextrin) is removed from the blood stream as a particle by the macrophages of the reticuloendothelial system and metabolized to replenish the body's iron stores of hemosiderin, ferritin and transferrin. The rate of removal from the blood stream is dependent on both the colloidal ferric hydroxide's particle size and composition.
Synthesis of Iron Carbohydrate Complexes
Iron carbohydrate complexes, such as iron sucrose, are composed of colloidal ferric hydroxide particles (i.e., cores) in complex with sucrose. These iron cores are prepared by the neutralization of ferric chloride with an alkali to a pH of 2. At this pH, the saturation of hydroxide ions induces the formation of colloidal ferric hydroxide, which after formation complexes in situ with a suitable carbohydrate, such as sucrose. The structure of the iron core follows classic coordination chemistry. The carbohydrate complexes with the iron core as its hydroxyl groups displace the water molecules bonded to the iron core's outer surface.
The bonding between the iron core and the carbohydrate is a non-covalent intermolecular force, such as the attraction of partial positive charges of the core's surface iron atoms to the negative dipole moments of the carbohydrate's hydroxyl groups.
Iron sucrose, for example, has a molecular weight (Mw) of about 34,000-60,000 Daltons and a molecular formula as follows:[Na2Fe5O8(OH)•3(H2O)]n.m(C12H22O11)where n is the degree of iron polymerization and m is the number of sucrose molecules (C12H22O11) in complex with the poly-nuclear polymerized iron core:[Na2Fe5O8(OH)•3(H2O)]n.
In solution, an equilibrium exists between a poly-nuclear polymerized iron core (Pn) and its solubilizing ligand (L):[Pn]+(m)[L]⇄[Pn]•m[L]
In order to assure a stable water-soluble iron complex, an excess amount of the solubilizing ligand is required and the equilibrium is as follows:[Pn]+(x)[L]→[Pn]•m[L]+(x−m)[L]A preferred method of synthesizing such iron carbohydrate complexes is described, for example, in published PCT Application WO 97/11711 (1997) by Lawrence et al.Evaluation of Iron Carbohydrate Homogeneity
Iron dextran complexes produced by the neutralization of ferric chloride in the presence of dextran have a similar structural formula, but differ in the degree of polymerization of the ferric hydroxide cores. See, for example, Lawrence (1998). The Lawrence paper also discusses methods to assess the homogeneity of particle sizes in an iron dextran complex by evaluating reduction degradation kinetics. After describing the evaluation of three iron dextran products from different manufacturers, the paper reports that there were marked differences among them in each of the physical and chemical parameters measured.
Determination of Bioequivalence of Iron Dextran Particles
As noted above, commercial iron supplement formulations are complex colloidal suspensions. For example, according to the USP Monograph for Iron Sucrose Injection by Luitpold Pharmaceuticals, Inc., to be published in the 2nd Supplement to the USP 25 in July/August 2002, such a formulation is pH controlled, and contains a controlled amount of particulate matter in addition to the iron and sucrose components. A comparison between commercial preparations of iron (III) dextrin complexes was reported by Erni, et al., “Chemical Characterization of Iron (III)-Hydroxide-Dextrin Complexes” Arzneim.-Forsch./Drug Res. 34(11):1555-1559 (1984). The paper noted that hydrolysis products of iron (III) may differ enormously in their structural, morphological, and chemical properties depending on the conditions under which they are formed and other factors. Attention was drawn to the nature of such hydrolysis products rather than the oxidation state of the iron—that is, iron (II) as compared with iron (III).
Erni et al. discusses the kinetic analysis of iron (III) reduction and relates it to the distribution of particle sizes and a range of surface to volume ratios in monodisperse as compared with polydisperse systems. See, for example, Sections 2.3 and 3.2 at pages 1556-57. Ascorbic acid, citric acid, phosphoric acid and sorbitol are a few of the reducing agents utilized by Erni, et al. Bioavailability in the context of oral preparations is also discussed; however, notably, these authors conclude that chemical tests alone will not allow for the prediction of bioavailability because they do not simulate the complex chemical environment of the intestine (See page 1559).
Other known processes have utilized the molecular weight distribution of a complex to correlate its bioavailability. However, this kind of distribution appears to vary dependent on the method, protocol and standards used in the molecular weight analysis. See, for instance, PCT WO97/11711 to Lawrence, et al.
General guidance on in vitro testing for immediate release solid oral dosage forms by dissolution testing is provided by the FDA at http://www.fda.gov/cder/guidance/1713bp1.pdf, and is entitled “Guidance for Industry: Dissolution Testing of Intermediate Release Solid Oral Dosage Forms.”
Thus, while an evaluation of the particle size distribution of iron-containing complexes has been reported, based upon their reduction degradation kinetics, the literature does not appear to have identified any particular correlation between the distribution of particle size and bioequivalence. Indeed, specific kinetic parameters, such as T75, have not heretofore been defined and associated with bioequivalence. What has been needed, therefore, is an accurate, inexpensive method for measuring reliably and consistently the bioequivalence of iron-containing compositions, as well as a quality control standard for so doing. Such a method would also permit the optimization of iron supplement formulations and the comparison of batches in production.